Bone Marrow Transplantation
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February 2000, Volume 25, Number 4, Pages 401-404
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Thalassaemia
Long-term survival of ex-thalassemic patients with persistent mixed chimerism after bone marrow transplantation
M Andreani, S Nesci, G Lucarelli, P Tonucci, S Rapa, E Angelucci, B Persini, F Agostinelli, M Donati and M Manna

Divisione di Ematologia e Centro Trapianto di Midollo Osseo, Ospedale di Muraglia, Pesaro, Italy

Correspondence to: Dr M Andreani, Divisione di Ematologia e Centro Trapianto di Midollo Osseo, Ospedale di Muraglia, Via Lombroso 19, 61100 Pesaro, Italy

Abstract

Twenty-six transplanted thalassemic patients out of 295 analyzed, showed the presence of persistent mixed chimerism, over a period of time varying between 2 and 11 years after BMT. Despite the presence of large numbers of residual host cells, these transplanted thalassemic patients no longer require red blood cell transfusions and have a functional graft, producing sufficient levels of hemoglobin A ranging from 8.3-14.7 g/dl. These ex-thalassemic patients with persistent mixed chimerism, although they did not achieve complete donor engraftment are no longer exposed to the risk of graft rejection. The mechanisms underlying this apparent state of tolerance or education in these patients are at the present time unknown. However, these observations may be useful for physicians involved in defining optimal strategies for clinical gene therapy, in uterohematopoietic stem cell transplantation and adoption of less toxic conditioning regimens in mini-transplantation. Bone Marrow Transplantation (2000) 25, 401-404.

Keywords

persistent mixed chimerism after BMT

In the early days of allogeneic bone marrow transplantation it was thought that ablation of all of the host stem cells was required to establish conditions for complete marrow engraftment of donor cells, ie complete chimerism (CC). Persistence of some host cells in the marrow along with donor cells, ie mixed chimerism (MC), was thought to presage rejection of the donor graft.1

However, we have observed that MC is not unusual in our group of transplanted thalassemics that now numbers almost 900. While MC occasionally evolves into graft rejection, other patients seem to move into a state of persistent, stable MC where their Hb levels are generally sufficient to allow good quality of life without any red blood cell (RBC) transfusion support.2 These observations led us to analyze MC in a group of 295 ex-thalassemic patients after bone marrow transplantation, all with a minimum follow-up of 2 years, in order to determine the incidence of MC at different periods after BMT and follow the evolution of MC over the time.

Some of 26 patients with persistent MC had a proportion of donor engrafted cells no more than 20-30%, suggesting that low numbers of donor erythroid precursor cells are sufficient to produce high levels of beta-globin chain synthesis and hemoglobin.3,4

These observations make possible the opportunity of investigating if the goal of 'cure' of thalassemic patients, aiming for establishing persistent MC rather than CC using less toxic myeloablative programs and consequently less regimen-related toxicity (RRT) may be achieved.5,6,7

Patients and materials

Patients

Among 824 patients transplanted in the BMT Unit of Pesaro from an HLA-identical sibling for homozygous beta-thalassemia, we selected 295 patients with 2 or more years of follow-up. Observations started from patient UPN 322, and were consecutive from patient UPN 788 to patient UPN 1257. We excluded from the study patients that had died, patients that never showed signs of engraftment and patients that did not return to our center for periodic clinical evaluation. Presence of donor cells in the recipient marrow or peripheral blood has been assessed at 2, 12 and 24 months and at every further clinical evaluation after BMT. Patients from all three classes of risk, categorized according to established criteria3 were included. One hundred and fifty-five patients were class 1 and 2, all treated with BU14 and CY200, 140 patients were class 3, treated with several different conditioning regimens with CY reduced to 120-160 mg/kg.

DNA extraction and restriction fragment length polymorphism analysis (RFLP)

High molecular weight DNA was extracted from peripheral blood (PB) or bone marrow (BM) using standard methods or from BFU-E agar colonies with intragene matrix (Biorad, Hercules, CA, USA). Southern blot analyses were performed with variable number tandem repeats (VNTR) probes as previously described.8 Calibration of the proportions of radioactive signals obtained was performed using 10%, 25%, 50%, 75% and 90% mixtures of two individuals with informative VNTR patterns. As already described, DNA dilution experiments allowed us to grade the amount of residual host cells (RHCs) present in the recipients into three different levels: MC level 1, when the RHCs were lower than 10%, MC level 2, when the RHCs ranged between 10 and 25% and MC level 3, when there were more than 25% RHCs.8

FISH analysis

Fluorescence in situ hybridization (FISH) was performed on PB and BM or on cells recovered from agar cultures when host and donor were sex mismatched. A biotinylated pY3.4 probe complementary to a part of the Y chromosome was used as reported in accordance with the manufacturer's instructions (Oncor, Gaithersburg, MD, USA).2 Positive and negative controls were included in each test.

Polymerase chain reaction (PCR)

To assess the chimeric state in poor DNA samples without usable markers, the highly polymorphic VNTR segments were amplified.2,9 Genomic regions thus examined with the specific set of primers as described, were locus D1S80 and locus D17S30. After amplification, 10% ultra-thin layer polyacrylamide gels were established for the electrophoresis and stained with silver. To evaluate the size-dependent efficacy of PCR amplification, calibration curves of donor recipient DNA mixtures were included.

BFU-E agar colonies

Erythroid burst-forming units (BFU-E) were grown in 0.3% agar in a humidified incubator gassed with CO2. Individual and pooled BFU-E were picked from culture dishes after 14 days.2

Results

Incidence of mixed chimerism after BMT

In this group of 295 patients (Table 1) with 2 or more years of post-transplant follow-up we observed an incidence of MC of 32.2% at 2 months after BMT. This proportion decreased to 12.8% 1 year after BMT and to 8.8% at 2 years after the transplant. Within this group of 295 patients, there were 155 class 1 or 2 patients, all treated with the same pretransplant conditioning regimen consisting of 14 mg/kg busulphan and 200 mg/kg cyclophosphamide. The 10% incidence of persistent MC 1 year and 12% 2 years after the transplant seen in this selected group of patients, homogeneous for the preparation to the transplant, was found to be similar to that observed in the remaining group of 140 patients.

Evolution of transient MC

None of the 200 ex-thalassemic patients with complete chimerism after BMT rejected their graft. Thirty-three of the 95 patients with MC observed within the first 2 months after BMT (34.7%) rejected the transplant.

Based on our prior classification scheme for MC (see Methods), we observed that 2 months after the transplant, 55 patients had MC level 1, 21 patients MC level 2 and 19 patients MC level 3. Table 2 shows the evolution of MC over time, starting with the second month engraftment status. Sixty percent of the patients with MC level 1 evolved towards complete chimerism, 12.7% rejected the transplant and 27.2% became persistent mixed chimeras. Thirty-eight percent of the patients with MC level 2 rejected the transplant, 42% became CC while 19% remained mixed chimeras. Eighteen of the 19 patients with MC level 3 rejected the transplant, while only one developed persistent PMC. The rejection rate and the incidence of MC were not associated with donor/recipient AB0 incompatibility, cell dose infused or cyclosporin treatment.

It is interesting to observe that while none of the 200 patients showing complete chimerism 2 months after BMT rejected the transplant, 3% of these patients at 2 years after the transplant showed the presence of persistent MC. The reason for this apparent contradiction could be the fact that early after BMT the amount of RHCs in these patients was below the limit of sensitivity of our tests that is about 1%. Data on the chimerism status at 12 months after BMT are also shown in Table 2.

Persistent mixed chimerism

Twenty-six ex-thalassemic patients after transplant maintained persistent PMC for a period of time from a minimum of 2 years to a maximum of 11 years (Table 3). They have all become transfusion independent with hemoglobin levels ranging from 8.3 g/dl to 14.7 g/dl. Eleven patients showed a persistent MC of level 3. We analyzed the donor/recipient proportion of erythroid precursors in these 11 patients at the time of the last observation distributed between 2 and 11 years after the transplant. As shown in Table 3, the proportion of singly picked BFU-E colonies confirmed the presence of relatively large numbers of recipient erythroid precursors in most of these patients. Figure 1 shows the donor marrow cells distribution over time in 11 patients with persistent MC level 3. It is interesting to observe that in the early post-transplant periods, the donor engrafted cell proportion decreased in some patients to levels usually predicting full rejection. We have observed, however, that change in the donor/recipient distribution over time is a characteristic of patients with persistent MC. This finding suggests that while high numbers of RHC in a recipient early after BMT reliably predicts rejection, the same proportion of RHC present after 2 years from BMT is consistent with a state of reciprocal tolerance between donor and recipient cells.

Discussion

Persistence of RHCs observed after marrow transplantation is not a rare event in thalassemia and is commonly referred to as MC.10,11 There is evidence that in some settings, MC is associated with an increased risk of graft failure and/or disease recurrence.1,2,12 Our data have shown that in thalassemic patients with transient MC, the probability of rejection is related to the number of RHCs present in the recipient in the early stage after BMT. We have shown that when there is the presence of more than 25% residual host cells within the first 2 months after BMT, the likelihood of graft rejection and return of thalassemia major is virtually certain (96%). In contrast, if at the same period of observation the proportion of RHCs is low, MC more likely evolves towards complete chimerism and the rejection rate is low.

Patients with transient MC characterized by the presence of high numbers of RHCs may in the future benefit from donor lymphocyte infusion (DLI) in order to reduce the risk of rejection, as proposed by others, in particular after the adoption of new therapeutic strategies, such as mini- transplants.5,13,14

Analysis of 295 ex-thalassemic patients after BMT has shown that the occurrence of transient MC does not necessarily lead to graft rejection. In fact, almost 9% of our patients achieved a state of stable MC wherein the number of donor marrow cells is sufficient to produce enough beta-globin synthesis with stable normal levels of hemoglobin in the peripheral blood so that transfusions to correct the anemia are no longer required.

The reasons why in some patients MC is transient, while in others the presence of donor and recipient cells remains persistent, are still unknown. We have observed, however, that after bone marrow transplantation some of the patients who became ex-thalassemic showed persistence of a high proportion of host type cells coexisting for as long as 11 years after the transplant with only a small proportion of donor cells. It is likely that a low number of donor cells is sufficient to correct the anemia and interrupt hypoxia-erythroid stimulation mediated through erythropoietin and therefore abolishes expansion of the residual host marrow.

These results, perhaps seen more clearly in the subgroup of 155 patients reflecting a single protocol in class 1 and 2 patients (data not shown), has potential importance for the future use of gene therapy, for in utero hematopoietic stem cell transplant or for adoption of less toxic conditioning regimens in mini-transplantation.6,7,15,16

It is our future aim to try to understand the mechanisms underlying this apparent state of tolerance or education in order to design programs that will allow us to produce it predictably, and hopefully with minimal toxicity.

Acknowledgements

We thank Dr Stanley Shrier for stimulating discussion and reviewing of the manuscript. This study was supported by the Berloni Foundation against Thalassemia, Pesaro, Italy.

References

1 Hill RS, Petersen FB, Storb R et al. Mixed hematologic chimerism after allogenic marrow transplantation for severe aplastic anemia is associated with a higher risk of graft rejection and a lessened incidence of acute graft-versus-host disease. Blood 1986; 67: 811-816, MEDLINE

2 Andreani M, Manna M, Lucarelli G et al. Persistence of mixed chimerism in patients transplanted for the treatment of thalassemia. Blood 1996; 87: 3494-3499, MEDLINE

3 Lucarelli G, Clift R, Galimberti M et al. Bone marrow transplantation in adult thalassemic patients. Blood 1999; 93: 1164-1167, MEDLINE

4 Lucarelli G, Galimberti M, Giardini C et al. Bone marrow transplantation in thalassemia. The experience of Pesaro. Ann NY Acad Sci 1998; 850: 270-275, MEDLINE

5 Sharabi Y, Abraham VS, Sykes M, Sachs D. Mixed allogeneic chimerism prepared by a non-myeloablative regimen: requirement for chimerism to maintain tolerance. Bone Marrow Transplant 1992; 9: 191-197, MEDLINE

6 Slavin S, Nagler A, Napastek E et al. Nonmyeloablative stem cell transplantation and cell therapy as an alternative to conventional bone marrow transplantation with lethal cytoreduction for the treatment of malignant and nonmalignant hematologic diseases. Blood 1998; 91: 756-763, MEDLINE

7 Dentamaro T, Cudillo L, Picardi A et al. Allogenic stem cell transplantation after non-myeloablative conditioning for poor risk patients with hematologic malignancies. Bone Marrow Transplant 1998; 21: (Suppl. 1) 115,

8 Nesci S, Manna M, Andreani M et al. Mixed chimerism in thalassemic patients after bone marrow transplantation. Bone Marrow Transplant 1992; 10: 143-147, MEDLINE

9 Smith AJ, Martin P, McFarland C et al. Evaluation of engraftment in the stem cell transplant setting by PCR DNA amplification and analysis of variable number tandem repeat loci. ASHI Quart 1998; 22: 3-9,

10 Petz LD, Yam P, Wallace RB et al. Mixed hematopoietic chimerism following bone marrow transplantation for hematologic malignancies. Blood 1987; 70: 1331-1337, MEDLINE

11 Bertheas MF, Lafage M, Levy P et al. Influence of mixed chimerism on the results of allogeneic bone marrow transplantation for leukemia. Blood 1991; 78: 3103-3107, MEDLINE

12 Huss R, Deeg H, Gooley T et al. Effect of mixed chimerism on graft-versus-host disease, disease recurrence and survival after HLA-identical marrow transplantation for aplasticanemia or chronic myelogenous leukemia. Bone MarrowTransplant 1996; 18: 767-776,

13 Aker M, Kapelushnik J, Pugatsch T et al. Donor lymphocyte infusions to displace residual host hematopoietic cells after allogeneic bone marrow transplantation for beta-thalassemia major. J Pediatr Hematol Oncol 1998; 20: 145-148, Article MEDLINE

14 Collins RH, Shpilberg O, Drobyski WR et al. Donor leukocyte infusions in 140 patients with relapsed malignancy after allogeneic bone marrow transplantation. J Clin Oncol 1997; 15: 433-444, MEDLINE

15 Elwood E, Larsen C, Maurer D et al. Microchimerism and rejection in clinical transplantation. Lancet 1997; 349: 1358-1360, Article MEDLINE

16 Flake A, Zanjani E. In utero hematopoietic stem cell transplantation. JAMA 1997; 278: 932-937, MEDLINE

Figures

Figure 1 Donor marrow cells distribution over the time in 11 patients with persistent MC level 3. PMC, persistent mixed chimerism.

Tables

Table 1  Characteristics of 295 patients included in the study

Table 2  Evolution of mixed chimerism from 2 to 24 months after BMT, based on the second month engraftment status

Table 3  Characteristics of 26 patients with persistent mixed chimerism

Received 20 July 1999; accepted 14 October 1999
February 2000, Volume 25, Number 4, Pages 401-404
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